Integrating bioprocess and metagenomics studies to enhance humic acid production from rice straw.

Rice straw burning annually (millions of tons) leads to greenhouse gas emissions, and an alternative solution is producing humic acid with high added-value. This study aimed to examine the influence of a microbial consortium and other additives (chicken manure, urea, olive mill waste, zeolite, and biochar) on the composting process of rice straw and the subsequent production of humic acid. Results showed that among the fungal species, Thermoascus aurantiacus exhibited the most prominent impact in expediting maturation and improving compost quality, and Bacillus subtilis was the most abundant bacterial species based on metagenomics analysis. The highest temperature, C/N ratio reduction, and amount of humic acid production (Respectively in lab 61 °C, 54.67%, 298 g kg-1 and in pilot level 65 °C, 72.11%, 310 g kg-1) were related to treatments containing these microorganisms and other additives except urea. Consequently, T. aurantiacus and B. subtilis can be employed on an industrial scale as compost additives to further elevate quality. Functional analysis showed that the bacterial enzymes in the treatments had the highest metabolic activities, including carbohydrate and amino acid metabolism compared to the control. The maximum enzymatic activities were in the thermophilic phase in treatments which were significantly higher than that in the control. The research emphasizes the importance of identifying and incorporating enzymatically active strains that are suitable for temperature conditions, alongside the native strains in decomposing materials. This strategy significantly improves the composting process and yields high-quality humic acid during the thermophilic phase.

Strain improvement of Trichoderma spp. through two-step protoplast fusion for cellulase production enhancement.

Fungal protoplast fusion is an approach to introduce novel characteristics into industrially important strains. Cellulases, essential enzymes with a wide range of biotechnological applications, are produced by many species of the filamentous fungi Trichoderma. In this study, a collection of 60 natural isolates were screened for Avicel and carboxymethyl cellulose degradation, and two cellulase producers of Trichoderma virens and Trichoderma harzianum were used for protoplast fusion. One of the resulting hybrids with improved cellulase activity, C1-3, was fused with the hyperproducer Trichoderma reesei Rut-C30. A new selected hybrid, F7, was increased in cellulase activity 1.8 and 5 times in comparison with Rut-C30 and C1-3, respectively. The increases in enzyme activity correlated with an upregulation of the cellulolytic genes cbh1, cbh2, egl3, and bgl1 in the parents. The amount of mRNA of cbh1 and cbh2 in F7 resembled that of Rut-C30 while the bgl1 mRNA level was similar to that of C1-3. AFLP (amplified fragment length polymorphism) fingerprinting and GC-MS (gas chromatography - mass spectrometry) analysis represented variations in parental strains and fusants. In conclusion, the results demonstrate that a 3-interspecific hybrid strain was isolated, with improved characteristics for cellulase degradation and showing genetic polymorphisms and differences in the volatile profile, suggesting reorganizations at the genetic level.

Protein engineering of chit42 towards improvement of chitinase and antifungal activities.

The antagonism of Trichoderma strains usually correlates with the secretion of fungal cell wall degrading enzymes such as chitinases. Chitinase Chit42 is believed to play an important role in the biocontrol activity of Trichoderma strains as a biocontrol agent against phytopathogenic fungi. Chit42 lacks a chitin-binding domain (ChBD) which is involved in its binding activity to insoluble chitin. In this study, a chimeric chitinase with improved enzyme activity was produced by fusing a ChBD from T. atroviride chitinase 18-10 to Chit42. The improved chitinase containing a ChBD displayed a 1.7-fold higher specific activity than chit42. This increase suggests that the ChBD provides a strong binding capacity to insoluble chitin. Moreover, Chit42-ChBD transformants showed higher antifungal activity towards seven phytopathogenic fungal species.